1. Field of the Invention
The present invention relates to the detection, identification and monitoring of submicron size particles. More particularly, the invention pertains to apparatus and method for the automated detection, identification, and monitoring of submicron size particles. Preferably, the present invention provides for the sampling, detection and identification of viruses and virus-like agents (such as, for example, prions, viral subunits, viral cores of delipidated viruses, etc.) in bioaerosols and fluids.
2. Brief Description of the Related Art
Detection and identification of viruses without limiting the detection and identification to a particular family, genus and species and searching for viruses pathogenic to humans in a single environment is difficult.
The difficulty of detecting and monitoring a wide range of viruses also varies by environment, but perhaps a most troublesome environment involves combat conditions, such as a potential biological warfare (BW) threat environment. Notwithstanding the variation in virulence from virus to virus, in general the ingestion of 104 virions constitutes a significant threat to a soldier who breathes on the order of 1,000 liters (1 m3) of air per hour. Instruments are needed with sensitivities which enable detection of remote releases of biological agents in a field environment thereby providing early warning capabilities, allowing calculations for troop movements and wind patterns.
Additionally, it has been difficult to maintain a broad-spectrum system for the detection of viruses which are free from false negatives because of natural or artificial mutations. Consideration should be given to the high mutation rates of known viruses, the emergence of new viruses, such as the Ebola virus, and the potential for deliberate artificial mutations of viruses. Furthermore, there are virus-like infectious agents, such as prions, which are suspected of causing scrapie, xe2x80x9cmad-cow diseasexe2x80x9d and Creutzfeldt-Jakob disease. These prions possess no DNA or RNA, and can withstand 8 MRads of ionizing radiation before losing infectiousness. Other virus-like infectious agents, such as satellites, possess no proteins.
In the detection and monitoring of viruses recognition should be given to false positives associated with background materials. Background includes biological debris which obscures the detection of the viruses by registering as a virus when a sample is analyzed. Analysis of viruses requires a very high degree of purification of those viruses to overcome background loading in order to avoid false positives. For example, a BW virus may be buried within loadings of other microorganisms which form biological debris having loading on a magnitude of 1010 larger than the threshold loading for the targeted virus itself.
Although methods that culture viruses can often be used to increase the virus over background, culture methods may be too slow for effective viral BW detection; furthermore, some important viruses cannot be easily cultured.
As set forth in U.S. Pat. No. 6,051,189 assigned to the U.S. Government and herein incorporated by reference, viruses may also be extracted from an environment and concentrated to an extent that permits detection and monitoring of viruses, without culturing procedures. Generally, in the detection of small amounts of viruses in environmental or biological liquids, it is necessary to both enrich the concentration of viruses many orders of magnitude (i.e., greatly reduce the volume of liquid solubilizing the viruses) and accomplish removal of non-viral impurities. In the presence of non-viral impurities, even the most sensitive detection methods generally require virus concentrations on the order of 10 femtomoles/microliter or more in the sampled liquid to reliably detect the viruses.
Sampling for airborne viruses is generally accomplished by collecting airborne particles in liquid, using a process such as air scrubbing, or eluting from filter paper collectors into a liquid medium. Collection and subsequent separation and detection methods are affected by the adsorption of viruses into solids in aerosols and liquids.
In contrast, when sampling liquids for viruses, in many cases no special equipment or processes may be necessary in order to collect a sample; for example, in sampling blood and other body fluids for viruses, only a standard clinical hypodermic needle may be needed. For sampling of bodies of water or other conveniently accessible liquids, sample collection may not be an issue at all, and in such cases the term xe2x80x9ccollectorxe2x80x9d is often applied to what is, in reality, a virus extraction step (such as collection on a filter).
Rapid detection translates into protection for soldiers, more reliable and simplified strategic planning, and validation of other BW countermeasures. Previously known detection methods using biochemical reagents may often be impractical in the field, even for trained virologists. Additionally, reagent-intensive approaches, such as multiplex PCR, low-strigency nucleic acid hybridization, and polyclonal antibodies, may increase the incidence of false positives several hundred-fold, whether under highly idealized laboratory conditions or in the field. Additionally, the hypervariability, or rapid mutation, of viruses and emergence of new, uncatalogued viruses may preclude methods based on biochemical assays, such as PCR, immunoassay, and the like, from achieving broad-spectrum detection of all viruses regardless of identity, known or unknown, sequenced or unsequenced.
A system and method for detecting the presence of submicron sized particles in a sample taken from the environment includes a collecting means for collecting a sample from the environment; and a means for purifying and concentrating the submicron particles in a sample by purifying and concentrating the particles based on size. The purifying and concentrating means includes a means for connecting the collecting means to the purifying and concentrating means for transferring the sample from the collecting means to the means for purifying and concentrating the particles. The system also includes a means for detecting the purified and concentrated particles, wherein the detecting means comprises: an electrospray assembly, the assembly having an electrospray capillary which receives the output from the purifying and concentrating means, a differential mobility analyzer which receives the output from the capillary, and a condensation particle device for counting the number of particles that pass through the differential mobility analyzer.
The collecting means comprises an ultracentrifuge for density-gradient ultracentifugation so that the particles are banded according to density, or a collector having means for liquid scrubbing a collected fluid sample of aerosol and gaseous materials containing the particles and a means for reducing the size of solid materials in the fluid sample. The collecting means may also comprise a liquid sample collector. The collecting means is intended to collect a sample containing submicron size particles having a size from about 10 to about 350 nanometers and are selected from the group consisting of viruses, prions, macromolecules, proteins and satellites
The system also includes a means for detecting the purified and concentrated particles, wherein the detecting means comprises: an electrospray assembly, the assembly having an electrospray capillary which receives the output from the purifying and concentrating means; a differential mobility analyzer which receives the output from said capillary; and which receives the output from the capillary, and a condensation particle device for counting the number of particles that pass through the differential mobility analyzer. Automated control means can be utilized to control the flow of sample through the system.
The method for detecting the presence of submicron sized particles in a sample taken from the environment, includes the steps of collecting a sample from the environment, purifying and concentrating the submicron size particles in the sample based on size; and detecting the purified and concentrated particles with a detecting means comprising an electrospray assembly which has an electrospray capillary which receives the output from the purifying and concentrating means, a differential mobility analyzer which receives the output from the capillary, and a condensation particle device for counting the number of particles that pass through the differential mobility analyzer.
Accordingly, an object of the present invention is to detect known and unknown or viruses and virus-like submicron size particles which may be pathogenic to humans according to the size of the particles.
Another object of the present invention is to provide a method and apparatus for the efficient and rapid detection and identification of viruses which is based on the physical characteristics of viruses.
A further object of the present invention to provide an automated system for the detection and identification of submicron size viruses and virus-like agents.
These, together with still other objects of the invention, along with the various features which characterize the invention, are pointed out with particularity in the appended claims.